Expression profiling of mouse endometrial cancers microdissected from ethanol-fixed, paraffin-embedded tissues

Abstract

Expression-profiling studies have helped define genetic changes associated with carcinogenesis. Determining which alterations in gene expression are causally associated with cancer and which result from the general dysregulation in gene expression that is characteristic of malignancies remains a problem. Transcriptional profiling of early lesions (small cancers or precancers) holds promise for identifying biologically important changes in gene expression. There are, however, technical barriers to the study of small tumors. The total number of cells available for analysis is limiting. It is also often difficult to distinguish cancer cells from normal proliferating cells in frozen sections that are typically used as a source of RNA. Here we describe an ethanol fixation and paraffin-embedding protocol that preserves tissue architecture and cellular morphology of the mouse endometrium, and allows for the recovery of high-quality RNA from microdissected cells. We performed GeneChip expression profiling using RNA from 800 to 4400 cells microdissected from ethanol-fixed, paraffin-embedded uteri. Endometrial adenocarcinomas exhibited changes in the levels of a number of messages known to be abnormally expressed in cancer, and differential expression of additional transcripts not previously implicated in carcinogenesis. We confirmed increased Amd1 expression in RNAs from mouse endometrial carcinomas that were hybridized to GeneChips and validated overexpression of this transcript in additional tumors.

Figure 1.

Representative examples of mouse uterine sections stained with H&E. A: Formalin-fixed normal mouse endometrium; C: ethanol-fixed normal mouse endometrium. E: Section through an ethanol-fixed uterus containing endometrial adenocarcinoma. A, C, and E: Low magnification; B, D, and F: high magnification. Note the good preservation of tissue architecture and cellular morphology in ethanol-fixed tissues. Scale bars, 100 μm.

Figure 2.

Qualitative and quantitative assessment of RNA from microdissected tissues. All tissues were fixed in ethanol and embedded in paraffin. Representative examples of Agilent LabChip and mini formaldehyde gel analysis are shown in A and B, respectively. Lanes 1 and 6: High-quality RNA standards (lane 1, 45 ng total RNA; lane 6, 1 ng total RNA). Lanes 3, 5, and 8: High-quality RNA with a 28S/18S rRNA ratio of ∼2 (lane 3, 5.16 ng total RNA; lane 5, 5.46 ng total RNA; lane 8, 1 ng total RNA). Lanes 2, 4, and 9: Semidegraded RNA. Note the 28S/18S rRNA ratio of <2 in lane 2, and the absence of a 28S rRNA band in lanes 4 and 9 (lane 2, 19.26 ng total RNA; lane 4, 14.4 ng total RNA; lane 9, 2 ng total RNA). Lane 7: Completely degraded RNA.

Figure 3.

Hybridization signals of amplification controls and visual representation of the GeneChip signals for 38 transcripts overrepresented in mouse endometrial cancer. A: Logarithmic (log₁₀) plot of the preamplification copy number of B. subtillis transcripts versus the scaled GeneChip signals of these control transcripts in each sample after two rounds of linear amplification, GeneChip hybridization, and signal scaling. The signals for the trp and thr transcripts show a linear correlation (black dotted line), while the scaled signals for the phe and lys messages appear to diverge from the linear plot (red dashed curve). B: A heat map of the 38 annotated transcripts that were overexpressed in all five cancers compared to both normal aRNA specimens that were hybridized to GeneChips. Red indicates an increase in signal intensity and green signifies a decrease in signal levels, both compared to the overall mean signal intensity for each transcript across all GeneChips (black). Colored boxes denote biological function(s) and/or involvement in oncogenesis.

Figure 4.

Validation of increased expression of Amd1 in mouse endometrial cancer. Quantitative, real-time PCR verifying overexpression of Amd1 in the five cancer RNA samples hybridized to GeneChips (Ca pools 1 and 2, and Ca A, Ca B, and Ca C), and validating increased expression of Amd1 in additional cancers (Ca D and Ca F). The average fold increase in Amd1 levels in specimens Ca A to Ca F (electronic pool represented by the average column) is within the range of fold increases seen for Ca pools 1 and 2 (biological pools). The fold increases were obtained by comparing Amd1 levels in each cancer to transcript levels in uninvolved normal endometrium from the same animal (see Materials and Methods).